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Table 1 Summary of the current published protocols for vascularized hPSC-derived organoids

From: The endothelium, a key actor in organ development and hPSC-derived organoid vascularization

Organoid ECs Vascularization strategy Endothelial characterization (Phenotype) FBS Ex. VEGF Commentaries Ref
Pancreas* HUVEC Coculture of HUVEC, MSCs and hPSC-derived PP progenitors.
MSC-driven condensation on Matrigel.
• Endothelial networks
• IF: CD31
In vivo: Anastomosis of human CD31+ networks with mouse blood vessels
+ + Initial % EC: 36%
In vivo reperfusion and non-leaky vessels
[14, 63]
HUVEC Coculture of HUVEC and hPSC-derived PP progenitors on a hydrogel (“amikagel”). • IF: vWF + Initial % EC: 50%
Final % EC: 45–50%
[65]
Liver** HUVEC Coculture of HUVEC, MSCs and hPSC-derived hepatic progenitors.
MSC-driven condensation on Matrigel.
• Endothelial networks
• IF: CD31
In vivo: Anastomosis of human CD31+ networks with mouse blood vessels
+ + Failure of non-vascularized (in vitro) organoids to engraft.
Initial % EC: 11%
[67, 68]
hPSC-EC Codifferentiation of hPSC into hepatobiliary and endothelial lineage on Matrigel. • Endothelial networks
• IF and FACS: CD34, CD31
• IF: CD144, CD146
• mRNA expression: JAG1, NOTCH2, HES1
  [71]
HAMEC EB differentiation of hepatic cells in the presence of HAMEC. • Endothelial rosettes
• IF: CD31+
• mRNA expression: Factor VIII, vWF and other coagulation cascade and fibrinolysis genes
In vivo: Human CD31+ in the rat spleen without migration to the liver.
+ Only vascularized organoids supported a sustained production of albumin in vivo up to 14 days after transplantation.
Initial % EC: 30%
Initial % EC: 15%
[70]
HUVEC Coculture of HUVEC, MSCs and hiPSC-derived hepatic progenitors.
MSC-driven condensation on Matrigel.
• Endothelial networks
• IF: CD31
• RNA-seq analysis
• Protein analysis of the culture supernatants
+ + Initial % EC: 45%
Surface contact of HE-iPSCs with non-parenchymal cells (HUVECs and MSCs) is required for organoid morphogenesis.
[69]
hPSC-EC HAEC Coculture of HAEC or iEC, MSCs or iMSC and hiPSC-derived hepatic progenitors.
MSC-driven condensation on Matrigel.
• Endothelial networks
• FACS: CD34, CD31, CD144 at t0
• Proteomic analysis
+ + Initial % EC: 40%
Differences on the organoid metabolic rate and TGF-β and Wnt signaling pathways were observed depending on the source of EC and MSC used.
[73]
hPSC-EC Coculture of iEC, iMSCs and hiPSC-derived hepatic progenitors.
iMSC-driven condensation on Matrigel.
• Endothelial networks
• IF: CD144, CD31
• scRNA-seq analysis
In vivo: Anastomosis with host mouse blood vessels surrounded by pericytes
+ + Completely hiPSC-derived liver organoids outperformed their counterpart made of HUVEC and MSCs.
Inhibition of KDR decreased the number of endothelial networks and hampered hepatocyte maturation.
[24, 72]
Kidney hPSC-EC Co-differentiation of hiPSC into kidney and endothelial lineage and self-assembling on transwells. • Endothelial networks with lumen
• IF: KDR, CD31, SOX17
  [79, 80]
hPSC-EC HUVEC Co-differentiation of hiPSC into kidney and endothelial lineage.
Self-assembling in low-attachment plates and further culture on transwells.
• Endothelial networks with lumen
• IF: KDR, CD31, CD34
• scRNA-seq analysis: Different subsets of EC
In vivo: Perfused and fenestrated vessels
+ Maximum 3% of EC at d14 [39]
hPSC-EC Co-differentiation of human pluripotent epiblast spheroids into kidney and endothelial lineage on Matrigel/Collagen gels. • Endothelial cords around capsule and tubular structures
• IF: CD31, vWF
Organoids survived for up to 2 months. [78]
hPSC-EC, HUVECS, HNDFs, and adult GMECs Co-differentiation of hiPSC into MM and endothelial lineage in suspension.
From d11 to d14, organoids were culture on perfused chips.
• Endothelial networks with lumen
• IF and FACS: CD31, MCAM and KDR
• mRNA expression: CD31
+   [81]
Brain hPSC-EC EB co-differentiation of neural progenitors and endothelial cells. • Vascular-like structures at d30;
• IF: CD144, CD31, vWF, KDR and tight junctions: OCLN, αZO-1
• EM: Tight junctions
• mRNA expression: CD144, CD31, KDR, TEK, vWF, OCLN, CD34, CLDN5, OCLN, TJP1, ABCB1 and GLUT1
• In vitro perfusion with Dextran-FITC.
• TEER: 351 ± 10 Ω cm− 2
• scRNA-seq analysis
In vivo: Anastomosis of human CD31+ networks with mouse blood vessels
+ EC came from modified hiPSC expressing ETV2 from day 18 of differentiation.
Organoids survived up to 4 months and reached 3,5–4 mm of diameter.
Vascularized organoids displayed better neural differentiation and neural functions.
[85]
hPSC-EC EB co-differentiation of neural progenitors and endothelial cells in low-attachment plates and further, embedded in Matrigel droplets. • Tubule-like structures
• IF: CD31, vWF, CLDN5
• mRNA expression: ANGPT1, CD31
• RNA-seq analysis: 106 TJ-related genes and GPR124
+ + Organoids survived up to 4 months.
α-SMA pericytes surrounded CD31+ EC.
[84]
hPSC-EC Fusion of hPSC-derived cortical and vascular spheroids. • IF: CD31, CD144, Z0–1
• FACS: CD31
• mRNA expression: BCRP, PGP, GLUT-1
NPSC: EC: hMSC (1:2:3)
Organoids survived up to 2 months.
[83]
hPSC-EC Co-culture of d34-brain organoids with ECs in Matrigel. • Tubular structures
• IF: CD31
In vivo: human CD31+ vessels.
+ + Organoids survived up to 2 months.
Only in vitro vascularized organoids survived in vivo 2 weeks after transplantation.
[82]
  1. Organ, endothelial cell source, vascularization strategy, endothelial characterization, use of fetal bovine serum (FBS) and exogenous VEGF (Ex. VEGF) are described for every vhPSC-Orgs protocol. IF: Immunofluorescence. FACS: fluorescence activated cell sorting. scRNA-seq analysis: single cell RNA sequencing analysis. *For pancreas organoids, authors often refer to pancreatic islet organoids because of the missing exocrine compartment. ** Liver organoids are also known in most cases as liver buds (LB)